Transcriptional regulatory factor

ABSTRACT

BLAST search was done on the EST database by using various nucleotide sequences encoding known bromodomain motifs to discover several ESTs likely encoding bromodomain genes. Next, testicular cDNAs were PCR cloned by using primers designed based on the sequence of EST (W17142), which is one of the ESTs discovered above. By using the thus obtained PCR product as a probe, the testicular library was screened. The obtained cDNA clone was used as a probe to re-screen the testicular cDNA library, thereby successfully isolating a full-length cDNA corresponding to EST (W17142). The protein encoded by the thus isolated cDNA had, in addition to the bromodomain, several regions and domains conserved in transcription regulatory factors. Moreover, the protein interacted with proteins associated with the chromatin-mediated transcriptional regulatory mechanism and a transcription co-activator.

This application is a continuation-in-part of PCT/JP99/02340, filed Apr.30, 1999, and claims priority from Japanese Patent Application No.10/137631, filed Apr. 30, 1998.

TECHNICAL FIELD

This invention relates to a novel transcriptional regulatory factorcomprising bromodomains and the encoding gene.

BACKGROUND OF THE INVENTION

The bromodomain is a characteristic amino-acid motif seen intranscriptional regulatory factors and is believed to be involved in theinteractions with other transcriptional regulatory factors. Proteinscomprising the bromodomain, normally have one or two (Tamkun et al.(1992) Nuc. Acids Res. 20:2603; Haynes et al. (1992) Nuc. Acids Res. 20:2603), but as many as five (Nicolas et al. (1996) Gene 175(12):233-240)bromodomain motifs. This motif has been identified in a wide range ofanimals, for example, in the homeotic gene (Digan et al. (1986) Dev.Biol. 114:161-169; Tamkun et al. (1992) Cell 68: 561-572) of the fruitfly (Drosophila), in the transcriptional regulatory genes of yeasts(Winston et al. (1987) Genetics 115:649-656; Laurent et al. (1991) Proc.Nat. Acad. Sci. USA 88:2687-2691) and in mammals (Denis et al. (1996)Genes and Devel. 10:261-271; Yang et al. (1996) Nature 382:319-324).According to a recent report (Jeanmougin et al. (1997) Trends Biochem.Sci. 22:151-153), 37 bromodomain genes, including 13 human genes arerecorded in the database. In addition to the bromodomain motif of aminoacid residues 59-63, the sequences adjacent to the motif are alsostructurally conserved, and furthermore, 4 α-helixes (Z, A, B, and C)are reported to be coded within the long 110 amino acids.

When these bromodomain-comprising transcriptional regulatory factors arecompared, they all regulate signal-dependent transcription in activelyproliferating cells (Tamkun et al. (1992) Cell 68:561-572; Haynes et al.(1992) Nuc. Acids Res. 20:2603). This characteristic implies thatoncogenesis may occur when a gene encoding a bromodomain-containingprotein undergoes abnormal regulation. In reality, six bromodomain geneshave been experimentally proven to associate with oncogenesis. Three ofthese genes HRX/ALL-1 (Tkachuk et al. (1992) Cell 71:691-700\; Gu et al.(1992) Cell 71:701-708); TIF1 (Miki et al. (1991) Proc. Nat. Acad. Sci.USA 88:5167-5171; Le Douarin et al. (1995) EMBO J. 14:2020-2033) and CBP(Borrow et al. (1996) Nature Genet. 14:33-41) are linked with the genecleavage points in leukemia. All three of these proteins contain theC4HC3 (also called PHD/LAP/TRX) zinc-finger (Aasland et al. (1995)Trends Biochem. Sci. 20:56-59; Koken et al. (1995) CRAcad. Sci. III,318:733-739; Saha et al. (1995) Proc. Nat. Acad. Sci. USA 92:9737-9741).Also, there are findings that CBP/P300 interact with p53 (Gu et al.(1997) Nature 387:819-823; Lill et al. (1997) Nature 387:823-827) andother various transcriptional factors, suggesting that CBP and thehomologous gene P300 play a key-role in cancer.

The other three genes have been suggested to be linked with cancer invarious ways. BRG1 interacts with retinoblastoma protein RB (Dunaief etal. (1994) Cell 79:119-130), inducing formation of flat, growth-arrestedcells, and thereby showing a tumor-suppressive activity. RING3 has ahomology with the fruit fly (Drosophila) growth control protein fsh(Haynes et al. (1989) Dev. Biol. 134:246-257) and is a serine-threoninekinase having endonuclear autophosphorylation activity. This activityhas been reported to be linked to the growth phase of chronic and acutelymphocytic leukemia (Denis et al. (1996) Genes and Devel. 10:261-271).As for P/CAF, it has been reported to inhibit the interaction betweenE1A and p300/CBP (Yang et al. (1996) Nature 382:319-324). When P/CAF isexogenously expressed on HeLa cells, the cell cycle is inhibited. Thisis believed to be due to the disruption of the transcriptionalregulation of E1A by the binding of P/CAF to p300/CBP. Similar top300/CBP (Bannister and Kouzarides (1996) Nature 384:641-643), P/CAF hasbeen reported to contain histone acetyl-transferase activity (Yang etal. (1996) Nature 382:319-324).

Thus, regulatory abnormalities of transcriptional regulatory factorscomprising bromodomains are envisaged to be closely associated withvarious diseases, particularly, cancer and othercell-proliferation-linked diseases. Hence, attention has been focused ontranscriptional regulatory factors comprising bromodomains in the recentyears as novel targets for the treatment of cancer and othercell-proliferation-linked diseases.

SUMMARY OF THE INVENTION

The present invention provides a novel transcriptional regulatory factorcomprising bromodomains, the encoding gene, a method of production, anda screening method for a drug-candidate compound that utilizes theprotein and the gene of the present invention.

In order to solve the above-mentioned problems, EST databases were BLASTsearched using various nucleotide sequences encoding known bromodomainmotifs. As a result, several potential bromodomain-gene-encoding ESTswere found by the search using nucleotide sequence of Tetrahymenathermophila HAT A1 gene. One of these ESTs, the fetal lung cDNAlibrary-derived EST (W17142) was found to encode an unknown gene.Therefore, isolation of full-length cDNA of EST W17142 was initiated.Specifically, primers were designed based on the EST W17142 sequence,and an amplification product was obtained by the polymerase chainreaction using testicular cDNA as the template. Then, the testicularcDNA library was screened using this amplification product as the probe,and a re-screening of the library was done using the cDNA clonecomprising the above-mentioned EST sequence, thereby successfullyisolating a full-length cDNA corresponding to EST W17142. By structuralanalysis of the protein encoded by the isolated cDNA, the presentInventors found that apart from the bromodomain, said protein hadseveral regions and domains conserved in transcriptional regulatoryfactors.

Also, they found that the protein encoded by the isolated cDNA interactswith hSNF2H and hSNF2L that are implicated in the series of processesrelated to the chromatin-mediated transcriptional regulatory mechanism,and also with the transcription co-activator NcoA-62/Skip, whichinteracts with the ligand-binding domains of various nuclear receptors(VDR, RAR) and the Ski viral oncoprotein.

The transcriptional regulatory factor and the encoding gene revealed bythe Inventors can be utilized for the screening of compounds inhibitingthe binding between said transcriptional regulatory factor and aninteracting factor, and compounds which regulate the binding activity.The compounds thus isolated are expected to be applied aspharmaceuticals.

Namely, the present invention relates to a novel transcriptionalregulatory factor comprising a bromodomain and the encoding gene, aswell as methods of production, and a screening method forrelated-factors and drug-candidate compounds that utilize the proteinand the gene of the present invention. Specifically, the presentinvention relates to:

1. a protein comprising the amino acid sequence of SEQ ID NO: 1 or 10;

2. a transcriptional regulatory factor comprising a bromodomain and theamino acid sequence of SEQ ID NO: 1 or 10, wherein one or more aminoacids are replaced, deleted, added, and/or inserted;

3. a protein comprising the amino acid sequence of SEQ ID NO: 1 or 10,wherein one or more amino acids are replaced, deleted, added, and/orinserted, and having an activity to bind to a protein selected from thegroup consisting of hSNF2H,hSNF2L,NCoA-62/Skip and homologues thereof;

4. a transcriptional regulatory factor comprising a bromodomain, andencoded by a DNA hybridizing with the DNA comprising the nucleotidesequence of SEQ ID NO:2 or 9;

5. a transcriptional regulatory factor encoded by a DNA hybridizing withthe DNA comprising the nucleotide sequence of SEQ ID NO:2 or 9, andhaving an activity to bind to a protein selected from the groupconsisting of hSNF2H, hSNF2L,NCoA-62/Skip and homologues thereof;

6. a DNA encoding the transcriptional regulatory factor of any one of(1) to (5);

7. the DNA of (6), which contains the coding region of the nucleotidesequence of SEQ ID NO:2 or 9;

8. a vector containing the DNA of (6) or (7);

9. a transformant carrying, in an expressible manner, the DNA of (6) or(7);

10. a method for producing the transcriptional regulatory factor of anyone of (1) to (5), the method comprising culturing the transformant of(9);

11. an antibody which binds to the transcriptional regulatory factor ofany one of (1) to (5);

12. a method for screening a compound having an activity to bind to thetranscriptional regulatory factor of any one of (1) to (5), the methodcomprising the steps of,

(a) exposing a test sample to said transcriptional regulatory factor,

(b) detecting the binding activity between the test sample and saidtranscriptional regulatory factor, and,

(c) selecting a compound having the binding activity to saidtranscriptional regulatory factor;

13. a method for screening a compound which promotes or inhibits thebinding between the transcriptional regulatory factor of any one of (1)to (5) and a protein selected from the group consisting of hSNF2H,hSNF2L, NCoA-62/Skip and homologues thereof, the method comprising thesteps of,

(a) exposing the transcriptional regulatory factor to hSNF2H, hSNF2L,NCoA-62/Skip or homologues thereof, in the presence of the test sample,

(b) detecting the binding activity between said transcriptionalregulatory factor and hSNF2H, hSNF2L, NCoA-62/Skip or homologuesthereof,

(c) selecting a compound which increases or decreases said bindingactivity when compared with the binding activity in the absence of thetest sample (control);

14. a compound which is obtainable by the method of (13), which inhibitsthe binding between the transcriptional regulatory factor of any one of(1) to (5) and a protein selected from the group consisting of hSNF2H,hSNF2L, NCoA-62/Skip and homologues thereof; and

15. a DNA comprising at least 15 nucleotides, which can specificallyhybridize with the DNA comprising the nucleotide sequence of SEQ ID NO:2or 9. The DNA can also be at least 351, 400, 450, 500, 700, 1000, 2200,2500, or 3000 bp in length.

Herein, “transcriptional regulatory factor” indicates a protein thatregulates gene expression. “Bromodomain” means, an amino acid motifassociated with protein-protein interactions conserved withintranscriptional regulatory factors linked to signal-dependenttranscription.

The present invention relates to a transcriptional regulatory factorcomprising a bromodomain. The amino acid sequences of the protein named“TCoA1” included in the present invention, and its variant are shown inSEQ ID NO: 1 and SEQ ID NO: 10, respectively, and the nucleotidesequences of their cDNA in SEQ ID NO:2 and SEQ ID NO:9, respectively(unless otherwise noted, these will be grouped as “TCoA1”, hereafter).“TCoA1” is most deeply associated with the presumed proteins of nematode(C. elegans) chromosome III genes F26H11.2, F26H11.3a and F26H11.3b(Wilson et al. (1994) Nature 368:32-38), the function of which areunknown and which were identified by the genomic sequence of one cosmidF26H11. When the amino acid sequence of these two proteins—the presumednematode protein and the “TCoA1” protein—are compared, although thedomain configurations are different, they are extremely alike.

Like many bromodomain proteins, “TCoA1” has one bromodomain. Beingstructurally similar to the TIF family, GCN5 and P/CAF, this bromodomainis situated close to the carboxyl-terminus (Jeanmougin et al. (1997)Trends Biochem. Sci. 22:151-153). Like other bromodomain proteins,“TCoA1” has a C4HC3 zinc-finger. The combination of the bromodomain andthe zinc-finger has been discovered frequently in the gene cleavagepoints in several leukemia, so far (Tkachuk et al. (1992) Cell71:691-700; Gu et al. (1992) Cell 71: 701-708; Miki et al. (1991) Proc.Nat. Acad. Sci. USA 88:5167-5171; Le Douarin et al. (1995) EMBO J.14:2020-2033; Borrow et al. (1996) Nature Genet. 14:33-41). Therefore,“TCoA1” is a candidate cleavage gene associated with chromosome no. 17q23.

“TCoA1” has numerous nuclear transport signal motifs. This indicatesthat “TCoA1” protein is located within the nucleus. Like otherbromodomain proteins, “TCoA1” has a is LXXLL motif series that likelydetermines the site of interaction with nuclear receptors (Heery et al.(1997) Trends Biochem. Sci. 22:151-153; Torchia et al. (1997) Nature387:677-684). The possibility that it interacts with the receptor boundto a ligand via the LXXLL domain indicates that “TCoA1” functions as atranscriptional co-activator. In the carboxyl terminus of “TCoA1”, aglutamine-rich domain is located spanning a very large region.Glutamine-rich domains have been identified in many transcriptionalregulatory factors including bromodomain-containing proteins likep300/CBP (Shikama et al. (1997) Trends in Cell Biol. 2:230-236) and fshprotein of fruit fly (Drosophila) (Haynes et al. (1989) Dev. Biol.134:246-257). These acidic regions have been predicted to be associatedwith the protein-protein interactions that determine the function as anactive substance (Courey et al. (1989) Cell 59:827-836).

“TCoA1” protein has many common characteristics with other bromodomainproteins known to be linked to cell-proliferation-linked diseases suchas cancer. Therefore, “TCoA1” protein may also be linked to cancer, andthus, the “TCoA1” protein and its gene, a compound that regulate thefunction of the “TCoA1” protein can be applied for the prevention andtreatment of cancer and other cell-proliferation-linked diseases.

Moreover, the fact that hSNF2H and hSNF2L, which interact with “TCoA1”,are involved in the series of processes related to thechromatin-mediated transcriptional regulatory mechanism, stronglyindicates that “TCoA1” is playing some sort of a role inchromatin-mediated transcriptional regulation. Therefore, it can beconceived that “TCoA1” is playing a major role as a protein thatintegrates transcriptional responses towards nuclear receptors byassociating with the chromatin reconstruction mechanism.

The transcriptional regulatory factor of the present invention can beprepared by methods known to one skilled in the art, as a recombinantprotein made using genetic engineering techniques, and also as a naturalprotein. For example, a recombinant protein can be prepared by insertingDNA encoding the protein of the present invention (for example, DNAcomprising the nucleotide sequence of SEQ ID NO:2 or 9) into a suitableexpression vector, introducing this into a host cell, and purifying theprotein from the resulting transformant. The natural protein can beacquired by preparing a column coupled with an antibody obtained byimmunizing a small animal with the recombinant protein, and performingaffinity chromatography for extracts of tissues or cells (for example,testis, tumor cells, etc.) expressing high levels of the transcriptionalregulatory factor of the present invention.

Also, this invention features a transcriptional regulatory factor, whichis functionally equivalent to the “TCoA1” protein (SEQ ID NO: 1 or 10).This transcriptional regulatory factor includes, mutants of the “TCoA1”protein (SEQ ID NO: 1 or 10) and “TCoA1” proteins obtained from variousliving organisms.

To isolate a protein functionally equivalent to a certain protein, themethod of inserting a mutation into the amino acids within the proteinis well known to one skilled in the art. In other words, for a personskilled in the art, the isolation of a transcriptional regulatory factorfunctionally equivalent to the “TCoA1” protein, is a standard procedurewhich can be done using, for example, the PCR-mediated,site-directed-mutation-induction system (GIBCO-BRL, Gaithersburg, Md.),oligonucleotide-mediated, sight-directed-mutagenesis (Kramer et al.(1987) Methods in Enzymol. 154:350-367) suitably replacing amino acidsthat do not influence the function of the “TCoA1” protein set forth inSEQ ID NO: 1 or 10. Mutations of amino acids can occur spontaneously aswell. The transcriptional regulatory factor of the invention includesthose comprising the amino acid sequence of “TCoA1” protein in SEQ IDNO:1 or 10 in which one or more amino acids have been replaced, deleted,added, and/or inserted, and have a binding-activity with hSNF2H, hSNF2Land NcoA-62/Skip, and those comprising the amino acid sequence of “TCoA1” protein in SEQ ID NO: 1 or 10 in which one or more amino acids havebeen replaced, deleted, added, and/or inserted, and comprise abromodomain.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. The substantially pure polypeptide is atleast 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Puritycan be measured by any appropriate standard method, for example, bycolumn chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

The number of amino acids that are mutated is not particularlyrestricted, as long as the function of the “TCoA1” protein ismaintained. Normally, it is within 50 amino acids, preferably within 30amino acids, more preferably within 10 amino acids and even morepreferably within 3 amino acids. The site of mutation may be any site,as long as the function of the “TCoA1” protein is maintained.

Proteins having amino acid sequences modified by deleting, adding and/orreplacing one or more amino acid residues of a certain amino acidsequence, have been known to retain the original biological activity(Mark et al., Proc. Natl. Acad. Sci. USA (1984) 81:5662-5666; Zoller etal. Nucleic Acids Research (1982) 10:6487-6500; Wang et al., Science224:1431-1433; Dalbadie-McFarland et al., Proc. Natl. Acad. Sci. USA(1982) 79:6409-6413).

As for the amino acid residue to be mutated, it is preferable to bemutated into a different amino acid in which the properties of the aminoacid side-chain are conserved. Examples of properties of amino acid sidechains are, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and aminoacids comprising the following side chains: an aliphatic side-chain (G,A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); asulfur atom containing side-chain (C, M); a carboxylic acid and amidecontaining side-chain (D, N, E, Q); a base containing side-chain (R, K,H); and an aromatic containing side-chain (H, F, Y, W). (The parentheticletters indicate the one-letter codes of amino acids). A “conservativeamino acid substitution is a replacement of one amino acid belonging toone of the above groups with another amino acid in the same group.

In the present invention, the protein having several deletions in theamino acid sequence of the “TCoA1” protein (SEQ ID NO:1 or 10) includesa partial peptide comprising binding-activity with hSNF2H, hSNF2L,NcoA-62/Skip or homologues thereof. As described in Example 6 (FIG. 5),the N-terminus of the “TCoA1” protein has a binding-activity withhSNF2H, hSNF2L, NcoA-62/Skip or homologues thereof. Peptides such asthese, inhibit the binding between “TCoA1” protein and the abovebinding-proteins in vivo, and thus can be used to inhibit the functionsof the “TCoA1” protein in vivo.

A fusion protein including the “TCoA1” protein can be given as anexample of a protein into which several amino acid residues have beenadded to the amino acid sequence of the “TCoA1” protein (SEQ ID NO:1 or10). Fusion proteins are, fusions of the “TCoA1” protein and otherpeptides or proteins, and are included in the present invention. Fusionproteins can be made by techniques well known to a person skilled in theart, such as by linking the DNA encoding the “TCoA1” protein of theinvention with DNA encoding other peptides or proteins, so as the framesmatch, inserting this into an expression vector and expressing it in ahost. There is no restriction as to the peptides or proteins fused tothe protein of the present invention.

Known peptides, for example, FLAG (Hopp et al., Biotechnology (1988)6:1204-1210), 6×His containing six His (histidine) residues, 10×His,Influenza agglutinin (HA), human c-myc fragment, VSP-GP fragment, p18HIVfragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag,α-tubulin fragment, B-tag, Protein C fragment, and such, can be used aspeptides that are fused to the protein of the present invention.Examples of proteins that are fused to protein of the invention are, GST(glutathione-S-transferase), Influenza agglutinin (HA), immunoglobulinconstant region, βgalactosidase, MBP (maltose-binding protein), andsuch.

Fusion proteins can be prepared by fusing commercially available DNAencoding these peptides or proteins with the DNA encoding the protein ofthe present invention and expressing the fused DNA prepared.

The hybridization technique (Sambrook et al., Molecular Cloning 2^(nd)ed. 9.47-9.58, Cold Spring Harbor Lab. press, 1989) is well known to oneskilled in the art as an alternative method for isolating a proteinfunctionally equivalent to a certain protein. In other words, for aperson skilled in the art, it is a general procedure to obtain atranscriptional regulatory factor functionally equivalent to the “TCoA1”protein, by isolating DNA having a high homology with the whole or partof the DNA encoding the “TCoA1” protein of SEQ ID NO:2 using thehybridization technique. The transcriptional regulatory factor of thepresent invention, includes transcriptional regulatory factorscomprising bromodomains which are encoded by the DNA hybridizing withthe DNA encoding “TCoA1” protein of SEQ ID NO:2. Animals which can beused to isolate a functionally equivalent transcriptional regulatoryfactor are, apart from humans, for example, mice, rats, cattle, monkeysand pigs, but there are no restrictions to the animal used. Thestringency of hybridization is defined as equilibrium hybridizationunder the following conditions: 42° C., 2×SSC, 0.1% SDS (lowstringency); 50° C., 2×SSC, 0.1% SDS (medium stringency); and 65° C.,2×SSC, 0.1% SDS (high stringency). If washings are necessary to achieveequilibrium, the washings are performed with the hybridization solutionfor the particular stringency desired. In general, the higher thetemperature, the higher is the homology between two strands hybridizingat equilibrium. However, several factors other than temperature caninfluence the stringency of hybridization and one skilled in the art cansuitably select the factors to accomplish a similar stringency.

In place of hybridization, the gene amplification method using a primersynthesized based on the sequence information of the DNA sequence of SEQID NO:9 encoding the “TCoA1” protein, for example, the polymerase chainreaction (PCR) method can be utilized to isolate a DNA encoding atranscriptional regulatory factor functionally equivalent to the “TCoA1”protein.

Proteins encoded by the DNA isolated through the above hybridizationtechnique or gene amplification techniques, normally have a highhomology to the amino acid sequence of the “TCoA1” protein. “Highhomology” refers to, normally a homology of 40% or higher, preferably60% or higher, more preferably 80% or higher, even more preferably 95%or higher with the amino acid sequence of the “TCoA1” protein. Thehomology of a protein can be determined by following the algorithm in“Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983) 80,726-730”.

The “percent identity” of two amino acid sequences or of two nucleicacids is determined using the algorithm of Karlin and Altschul (Proc.Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlin andAltschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotidesearches are performed with the NBLAST program, score=100,wordlength=12. BLAST protein searches are performed with the XBLASTprogram, score=50, wordlength=3. Where gaps exist between two sequences,Gapped BLAST is utilized as described in Altschul et al. (Nucleic AcidsRes. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.

An “isolated nucleic acid” is a nucleic acid the structure of which isnot identical to that of any naturally occurring nucleic acid or to thatof any fragment of a naturally occurring genomic nucleic acid spanningmore than three separate genes. The term therefore covers, for example,(a) a DNA which has the sequence of part of a naturally occurringgenomic DNA molecule but is not flanked by both of the coding sequencesthat flank that part of the molecule in the genome of the organism inwhich it naturally occurs; (b) a nucleic acid incorporated into a vectoror into the genomic DNA of a prokaryote or eukaryote in a manner suchthat the resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion protein.Specifically excluded from this definition are nucleic acids present inmixtures of different (i) DNA molecules, (ii) transfected cells, or(iii) cell clones: e.g., as these occur in a DNA library such as a cDNAor genomic DNA library.

Transcriptional regulatory factors functionally equivalent to the“TCoA1” protein (SEQ ID NO: 1 or 10) isolated by the above hybridizationtechnique or gene amplification techniques include, those having abinding activity with hSNF2H, hSNF2L and NcoA-62/Skip, and a highhomology in the primary structure with the “TCoA1” protein (SEQ ID NO:1or 10), and those having the bromodomain, which is a motif thought to bevital to the function linked with cancer, and a high homology in theprimary structure with the “TCoA1” protein (SEQ ID NO:10).

Other than the bromodomain, these transcriptional regulatory factorsalso comprise sequences involved in the interactions with other proteins(for example, leucine-zipper, LXXLL motif), sequences involved in thebinding with DNA (for example, zinc-finger), and nuclear transportsignals.

The existence of the bromodomain within a protein can be determined bysearching the bromodomain motif PROSITE database on DNASIS (HitachiSoftware Engineering).

This invention also relates to a DNA encoding the above transcriptionalregulatory factor. There is no restriction as to the DNA of the presentinvention as long as it encodes the transcriptional regulatory factor ofthe invention, and includes cDNA, genomic DNA and chemically synthesizedDNA. Also as long as they can encode the protein of the invention, DNAscomprising arbitrary sequences based on the degeneracy of the geneticcode are also included. cDNA encoding the protein of the invention canbe prepared, for example, by preparing a primer based on nucleotideinformation (for example, SEQ ID NO:9) of DNA encoding thetranscriptional regulatory factor of the invention and performing plaquePCR (for example please refer, Affara NA et al. (1994) Genomics22:205-210). In the case of genomic DNA, preparation can be done forexample, by the method using commercially available “Qiagen genomic DNAkits” (Qiagen, Hilden, Germany). The nucleotide sequence of the DNAacquired can be decided by ordinary methods in the art by using, forexample, the commercially available “dye terminator sequencing kit”(Applied Biosystems). The DNA of the present invention, as stated later,can be utilized for the production of a recombinant protein and genetherapy.

The present invention also features a vector into which the DNA of thepresent invention has been inserted. There is no restriction as to thevector to which DNA is inserted, and various vectors such as those forexpressing the transcriptional regulatory factor of the presentinvention in vivo and those for preparing the recombinant protein can beused according to the objective. To express the transcriptionalregulatory factor of the present invention in vivo (especially for genetherapy), various viral vectors and non-viral vectors can be used.Examples of viral vectors are, adenovirus vectors (pAdexLcw) andretrovirus vectors (pZlPneo), etc. Cationic liposomes can be given asexamples of non-viral vectors. Expression vectors are especially usefulwhen using for the purpose of producing the transcriptional regulatoryfactor of the invention. For example, when using colibacili (E. coli)the “pREP4” (Qiagen, Hilden, Germany) and such vectors, when using yeast“SP-Q01” (Stratagene, La Jolla, Calif.) and such, when using insectcells “Bac-to-Bac baculovirus expression system” (GIBCO-BRL,Gaithersburg, Md.) are highly appropriate, but there is no restriction.Also, when using mammalian cells such as CHO cells, COS cells,NIH3T3cells, for example, the “LacSwitch II expression system(Stratagene, La Jolla, Calif.) is highly suitable, but there is norestriction. Insertion of the DNA of the present invention into a vectorcan be done using ordinary methods in the art.

The present invention also refers to a transformant, carrying, in anexpressible manner, the DNA of the present invention. The transformantof the present invention includes, those carrying the above-mentionedvector into which DNA of the present invention has been inserted, andthose having host genomes into which the DNA of the present inventionhas been integrated. As long as the DNA of the present invention ismaintained in an expressible manner, no distinction is made as to theform of existence of the transformants. There is no particularrestriction as to the cells into which the vector is inserted. Forexample, when using for the purpose of gene therapy, various cells canbe used as target cells according to the type of disease. Also, when thepurpose is to produce the transcriptional regulatory factor of thepresent invention, for example, E. coli, yeast, animal cells and insectcells can be used as hosts. Introduction of a vector into a cell can bedone using known methods such as electroporation and calcium phosphatemethod.

Common methods applied in the art may be used to isolate and purify saidrecombinant protein from the transformant made for the production ofrecombinant proteins. For example, after collecting the transformant andobtaining the extracts, the objective protein can be purified andprepared by, ion exchange chromatography, reverse phase chromatography,gel filtration, or affinity chromatography where an antibody against theprotein of the present invention has been immobilized in the column, orby combining several of these columns.

Also when the protein of the present invention is expressed within hostcells (for example, animal cells and E. coli) as a fusion protein withglutathione-S-transferase protein or as a recombinant proteinsupplemented with multiple histidines, the expressed recombinant proteincan be purified using a glutathione column or nickel column. Afterpurifying the fusion protein, it is also possible to exclude regionsother than the objective protein by cutting with thrombin or factor-Xaas required.

The present invention also features an antibody binding to thetranscriptional regulatory factor of the invention. There is noparticular restriction as to the form of the antibody of the presentinvention and include, apart from polyclonal antibodies, monoclonalantibodies as well. The antiserum obtained by immunizing animals such asrabbits with the transcriptional regulatory factor of the presentinvention, polyclonal and monoclonal antibodies of all classes,humanized antibodies made by genetic engineering, human antibodies, arealso included. The antibodies of the present invention can be preparedby the following methods. Polyclonal antibodies can be made by,obtaining the serum of small animals such as rabbits immunized with thetranscriptional regulatory factor of the present invention, attaining afraction recognizing only the transcriptional regulatory factor of theinvention by an affinity column coupled with the protein of the presentinvention, and purifying immunoglobulin G or M from this fraction by aprotein G or protein A column. Monoclonal antibodies can be made byimmunizing small animals such as mice with the transcriptionalregulatory factor of the present invention, excising the spleen from theanimal, homogenizing the organ into cells, fusing the cells with mousemyeloma cells using a reagent such as polyethylene glycol, selectingclones that produce antibodies against the transcriptional regulatoryfactor of the invention from the fused cells (hybridomas), transplantingthe obtained hybridomas into the abdominal cavity of a mouse, andextracting ascites. The obtained monoclonal antibodies can be purifiedby, for example, ammonium sulfate precipitation, protein A or protein Gcolumn, DEAE ion exchange chromatography, or an affinity column to whichthe transcriptional regulatory factor of the present invention iscoupled. The antibody of the invention can be used for purifying anddetecting the transcriptional regulatory factor of the invention. It canalso be used as a pharmaceutical drug to inhibit the function of thepresent transcriptional regulatory factor. When using the antibody as adrug, in the view-point of immunogenicity, human antibodies or humanizedantibodies are effective. The human antibodies or humanized antibodiescan be prepared by methods commonly known to one skilled in the art. Forexample, human antibodies can be made by, immunizing a mouse whoseimmune system has been changed to that of humans, with thetranscriptional regulatory factor of the invention. Also, humanizedantibodies can be prepared by, for example, cloning the antibody genefrom monoclonal antibody producing cells and using the CDR graft methodwhich transplants the antigen-recognition site of the gene into a knownhuman antibody.

The present invention also relates to a method for screening a compoundthat binds to the transcriptional regulatory factor of the invention.The screening method of the invention includes the steps of, (a)exposing a test sample to the transcriptional regulatory factor of theinvention, (b) detecting the binding activity between the test sampleand the transcriptional regulatory factor of the invention, and (c)selecting a compound having an activity to bind to the transcriptionalregulatory factor of the invention. Any test sample can be used for thescreening without particular restrictions. Examples are, cell extracts,culture supernatants, synthetic low molecular weight compound libraries,purified proteins, expression products of gene libraries, syntheticpeptide libraries, and so on.

Isolation of a compound that binds to the transcriptional regulatoryfactor using said transcriptional regulatory factor can be done usingmethods commonly known to one skilled in the art. The screening of aprotein which binds to the transcriptional regulatory factor of theinvention can be done by, for example, creating a cDNA library fromtissues or cells (for example, testis tissue cells and tumor cell lines)expected to express a protein binding to the transcriptional regulatoryfactor of the invention using a phage vector (λgt11 and Zap, etc.),expressing this cDNA library on LB-agarose, fixing the expressedproteins on the filter, biotin-labeling the transcriptional regulatoryfactor of the invention or purifying it as a fusion protein with GSTprotein, reacting this with the above-described filter, and detectingplaques expressing the binding proteins using streptavidin or anti-GSTantibody (West Western Blotting method) (Skolnik et al. (1991) Cloningof PI3 kinase-associated p85 utilizing a novel method forexpression/cloning of target proteins for receptor tyrosine kinases,Cell 65:83-90). The screening of a protein binding to thetranscriptional regulatory factor of the invention or its gene, can alsobe done by following “the two-hybrid system” (“MATCHMAKER Two-hybridSystem”, “Mammalian MATCHMAKER Two-Hybrid Assay Kit”, “MATCHMAKEROne-Hybrid System” (Clontech), “HybriZAP Two-Hybrid Vector System”(Stratagene), or Reference—“Dalton S, and Treisman R (1992)Characterization of SAP-1, a protein recruited by serum response factorto the c-fos serum response element. Cell 68, 597-612”). In thetwo-hybrid system, the transcriptional regulatory factor of theinvention is fused to the SRF-binding region or GAL4-binding region andexpressed in yeast cells. A cDNA library, is prepared from cellsexpected to express a protein binding to the transcriptional regulatoryfactor of the invention, in a way that the library is expressed in theform of being fused to the VP16 or GAL4 transcriptional activationregion. The cDNA library is then introduced into the above yeast cellsand the cDNA derived from the library is isolated from the positiveclones detected (when a protein binding to the transcriptionalregulatory factor of the invention is expressed in yeast cells, thebinding of the two activates a reporter gene making positive clonesdetectable). A protein binding to the transcriptional regulatory factorof the invention can be recovered by, introducing the cDNA isolatedabove to E. coli and expressing the protein encoded by said cDNA.

Also, a protein binding to the transcriptional regulatory factor of theinvention can be screened by, applying the culture supernatants or cellextracts of cells expected to express a protein binding to thetranscriptional regulatory factor of the invention onto an affinitycolumn in which the protein of the invention is immobilized andpurifying the protein that binds specifically to the column.

The method of screening molecules that bind when the immobilizedtranscriptional regulatory factor of the invention is exposed tosynthetic chemical compounds, or natural substance banks, or a randomphage peptide display library, or the method of screening usinghigh-throughput based on combinatorial chemistry techniques (Wrighton etal., Small peptides as potent mimetics of the protein hormoneerythropoietin, Science (UNITED STATES) (1996), 273:458-464; Verdine G.L., The combinatorial chemistry of nature, Nature (ENGLAND) (1996)384:11-13; Hogan J. C., Jr., Directed combinatorial chemistry. Nature(ENGLAND) (1996) 384:17-19) to isolate low molecular weight compounds,proteins (or their genes) and peptides are methods well known to oneskilled in the art.

A biosensor using the surface plasmon resonance phenomenon may be usedas a mean for detecting or quantifying the bound compound in the presentinvention. When such a biosensor is used, the interaction between theprotein of the invention and a test compound can be observed real-timeas a surface plasmon resonance signal, using only a minute amount ofproteins without labeling (for example, BIAcore, Pharmacia). Therefore,it is possible to evaluate the binding between the transcriptionalregulatory factor of the invention and a test compound using a biosensorsuch as BIAcore.

The present invention also relates to a method for screening a compoundable to promote or inhibit the binding between the transcriptionalregulatory factor of the invention and an interacting-protein. Detectionof a binding between the TCoA1 protein and hSNF2H, hSNF2L, NCoA-62/Skipor homologues thereof enabled such a screening. This screening can bedone using the method comprising the steps of: (a) exposing thetranscriptional regulatory factor of the invention to hSNF2H, hSNF2L,NCoA-62/Skip or homologues thereof, under the presence of a test sample;(b) detecting the binding activity between the transcriptionalregulatory factor of the invention and hSNF2H, hSNF2L, NCoA-62/Skip orhomologues thereof; and (c) selecting a compound which decreases saidbinding-activity when compared with the assay in the absence of a testsample (control).

There are no particular restrictions as to the test sample used.Examples are, cell extracts, culture supernatants, libraries ofsynthetic low molecular weight compounds, purified proteins, expressionproducts of gene libraries, synthetic peptide libraries, etc. Thecompound isolated by the above-described screening of a protein bindingto the protein of the invention may also be used as a test sample.

The transcriptional regulatory factor of the invention used for thescreening may be a whole protein or a partial peptide comprising bindingregions with hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof. hSNF2H,hSNF2L, NCoA-62/Skip or homologues thereof used for the screening may bewhole proteins or partial peptides comprising binding regions with thetranscriptional regulatory factor of the invention.

The detection of the binding activity between the transcriptionalregulatory factor of the invention and hSNF2H, hSNF2L, NCoA-62/Skip orhomologues thereof, can be performed, for example, as follows.

A test sample and hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof isadded to the transcriptional regulatory factor of the inventionimmobilized on a microplate, reacted with a mouse or rabbit antibodyagainst hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof, furtherreacted with an anti-mouse or anti-rabbit antibody labeled withperoxidase, alkaline phosphatase and such, a labeled enzyme substrate isadded and the enzyme activity is measured. Compounds that show an enzymeactivity that is lower to or higher than that in the absence of a testsample, are selected. Thereby, compounds having an activity to promoteor inhibit the binding between the transcriptional regulatory factor ofthe invention and hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof areobtained.

This screening may be performed also by, using hSNF2H, hSNF2L,NCoA-62/Skip or homologues thereof as the immobilized protein, and thetranscriptional regulatory factor of the invention as the protein thatis added with the test sample.

Also, the transcriptional regulatory factor of the invention or hSNF2H,hSNF2L, NCoA-62/Skip or homologues thereof added together with the testsample may be directly labeled with peroxidase, or alkaline phosphatase,or used as a fusion protein with such enzymes. Compounds having anactivity that activates or inhibits the binding between thetranscriptional regulatory factor of the invention and hSNF2H, hSNF2L,NCoA-62/Skip or homologues thereof may also be selected by, expressingas fusion proteins with enzymes other than the above, such as,luciferase, β-galactosidase, or GFP protein and measuring the inhibitionor promotion of the enzyme activity by a test sample.

The mammalian two-hybrid system (Clontech, Palo Alto) can also be usedto screen a compound that promotes or inhibits the binding between thetranscriptional regulatory factor of the invention and aninteracting-protein. Namely, using the two-hybrid system, thetranscriptional regulatory factor of the invention and aninteracting-protein is expressed in mammalian cells, a test sample isadded to said mammalian cells, and then reporter-activity is measured.The detected reporter-activity is compared, and compounds that give avalue that is lower to or higher than the reporter-activity in theabsence of a test sample, are selected. Thus, a compound that promotesor inhibits the binding between the transcriptional regulatory factor ofthe invention and hSNF2H, hSNF2L, NCoA-62/Skip or homologues thereof canbe obtained.

A compound screened by the screening of the invention may be applied forthe prevention and treatment of cancer and othercell-proliferation-linked diseases. When using the isolated compound asa pharmaceutical for humans and other mammals, such as, mice, rats,guinea-pigs, rabbits, chicken, cats, dogs, sheep, pigs, monkeys,baboons, chimpanzees, the isolated compound can be directly administeredor can be formulated into a dosage form using known pharmaceuticalpreparation methods. For example, according to the need, the drugs canbe taken orally as sugar-coated tablets, capsules, elixirs andmicrocapsules or non-orally in the form of injections of sterilesolutions or suspensions with water or any other pharmaceuticallyacceptable liquid. For example, the compounds can be mixed withpharmacologically acceptable carriers or medium, specifically,sterilized water, physiological saline, plant-oil, emulsifiers,solvents, surfactants, stabilizers, flavoring agents, excipients,vehicles, preservatives and binders, in a unit dose form required forgenerally accepted drug implementation. The amount of active ingredientsin these preparations makes a suitable dosage within the indicated rangeacquirable.

Examples for additives which can be mixed to tablets and capsules are,binders such as gelatin, corn starch, tragacanth gum and arabic gum;excipients such as crystalline cellulose; swelling agents such as cornstarch, gelatin and alginic acid; lubricants such as magnesium stearate;sweeteners such as sucrose, lactose or saccharin; flavoring agents suchas peppermint, Gaultheria adenothrix oil and cherry. When the unitdosage form is a capsule, a liquid carrier, such as oil, can also beincluded in the above ingredients. Sterile composites for injections canbe formulated following normal drug implementations using vehicles suchas distilled water used for injections.

Physiological saline, glucose, and other isotonic liquids includingadjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodiumchloride, can be used as aqueous solutions for injections. These can beused in conjunction with suitable solubilizers, such as alcohol,specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, non-ionic surfactants, such as Polysorbate 80 (™)and HCO-50.

Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may beused in conjunction with benzyl benzoate or benzyl alcohol as asolubilizers; may be formulated with a buffer such as phosphate bufferand sodium acetate buffer; a pain-killer such as procaine hydrochloride;a stabilizer such as benzyl alcohol, phenol; and an anti-oxidant. Theprepared injection is filled into a suitable ampule.

Methods well known to one skilled in the art may be used to administer apharmaceutical compound to patients, for example as intraarterial,intravenous, percutaneous injections and also as intranasal,transbronchial, intramuscular or oral administrations. The dosage andmethod of administration vary according to the body-weight and age of apatient and the administration method but one skilled in the art cansuitably select them. If said compound is encodable by a DNA, said DNAcan be inserted into a vector for gene therapy and perform the therapy.The dosage and method of administration vary according to thebody-weight, age, and symptoms of a patient but one skilled in the artcan select them suitably.

For example, although there are some differences according to thesymptoms, the dose of a compound that binds with the transcriptionalregulatory factor of the present invention and regulates its activity isabout 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about50 mg per day and more preferably about 1.0 mg to about 20 mg per day,when administered orally to a normal adult (weight 60 kg).

When administering parenterally in the form of an injection to a normaladult (weight 60 kg), although there are some differences according tothe patient, target organ, symptoms and method of administration, it isconvenient to intravenously inject a dose of about 0.01 mg to about 30mg per day, preferably about 0.1 to about 20 mg per day and morepreferably about 0.1 to about 10 mg per day. Also, in the case of otheranimals too, it is possible to administer an amount converted to 60 kgof body-weight.

This invention also features a DNA containing at least 15 nucleotides,which can specifically hybridize with DNA encoding the “TCoA1” protein.The term “specifically hybridize” as used herein, indicates thatcross-hybridization does not occur significantly with DNA encoding otherproteins, in the above-mentioned hybridizing conditions, preferablyunder stringent hybridizing conditions. Such DNA includes, probes,primers, nucleotides and nucleotide derivatives (for example, antisenseoligonucleotides and ribozymes), which specifically hybridize with DNAencoding the protein of the invention or its complementary DNA.

The present invention includes an antisense oligonucleotide thathybridizes with any site within the nucleotide sequence of SEQ ID NO:2or 9. This antisense oligonucleotide is preferably that against the atleast 15 continuous nucleotides in the nucleotide sequence of SEQ IDNO:2 or 9. The above-mentioned antisense oligonucleotide, which containsan initiation codon in the above-mentioned at least 15 continuousnucleotides, is even more preferred.

Derivatives or modified products of antisense oligonucleotides can beused as antisense oligonucleotides. Examples of such modified productsare, lower alkyl phosphonate modifications such asmethyl-phosphonate-type or ethyl-phosphonate-type, phosphothioatemodifications and phosphoramidate modifications.

The term “antisense oligonucleotides” as used herein means, not onlythose in which the entire nucleotides corresponding to thoseconstituting a specified region of a DNA or mRNA are complementary, butalso those having a mismatch of one or more nucleotides, as long as DNAor mRNA and an oligonucleotide can specifically hybridize with thenucleotide sequence of SEQ ID NO:9.

Such DNAs are indicated as those having, in the “at least 15 continuousnucleotide sequence region”, a homology of at least 70% or higher,preferably at 80% or higher, more preferably 90% or higher, even morepreferably 95% or higher. The algorithm stated herein can be used todetermine homology. Such DNAs are useful as probes for the isolation ordetection of DNA encoding the protein of the invention as stated in alater example or as a primer used for amplifications.

The antisense oligonucleotide derivative of the present invention, actsupon cells producing the protein of the invention by binding to the DNAor mRNA encoding the protein and inhibits its transcription ortranslation, promotes the degradation of the mRNA, inhibiting theexpression of the protein of the invention resulting in the inhibitionof the protein's function.

The antisense oligonucleotide derivative of the present invention can bemade into an external preparation such as a liniment and a poultice bymixing with a suitable base material, which is inactive against thederivatives.

Also, as needed, the derivatives can be formulated into tablets,powders, granules, capsules, liposome capsules, injections, solutions,nose-drops and freeze-drying agents by adding excipients, isotonicagents, solubilizers, stabilizers, preservatives, pain-killers, andsuch. These can be prepared by following usual methods.

The antisense oligonucleotide derivative is given to the patient by,directly applying onto the ailing site or by injecting into a bloodvessel so that it will reach the site of ailment. An antisense-mountingmedium can also be used to increase durability andmembrane-permeability. Examples are, liposome, poly-L-lysine, lipid,cholesterol, lipofectin or derivatives of these.

The dosage of the antisense oligonucleotide derivative of the presentinvention can be adjusted suitably according to the patient's conditionand used in desired amounts. For example, a dose range of 0.1 to 100mg/kg, preferably 0.1 to 50 mg/kg can be administered.

The antisense oligonucleotide of the invention inhibits the expressionof the protein of the invention and thereby useful for suppressing thebiological activity of the protein of the invention. Also,expression-inhibitors comprising the antisense oligonucleotide of theinvention are useful in the point that they can inhibit the biologicalactivity of the protein of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the alignment of the domains identified in “TCoA1”. Thesymbols within the figure are shown below.

CH4C3: CH4C3 zinc-finger; bHLH: basic helix-loop-helix; Q-rich:glutamine-rich;

C2HC4: C2HC4 zinc-finger; BDM: bromodomain; ↑: LXXLL motif.

FIG. 2A shows the results of analysis of mono-chromosome hybrid cellpanel against chromosome no. 17 using nb15G and nb15H primers. Shows thehuman chromosome including each hybrid. The product of 133 bp wasspecifically amplified in GM10498 cell-system, which is amono-chromosome of human chromosome no. 17.

FIG. 2B shows the result of GeneBridge 4 radiation hybrid panel analysisby which the location of “TCoA1” was determined on chromosome no. 17.

FIG. 3 shows the electrophoretic pattern of the “TCoA1” expression innormal human tissues as detected by northern-blot analysis. “TCoA1” wasused as the probe when hybridizing the filter in “A”, and actin was usedin “B”. The right side of the figure shows markers.

FIG. 4 shows the results obtained by using the mammalian two-hybridanalysis system detecting the interaction between “TCoA1” and hSNF2H,hSNF2L, and NcoA-62/Skip.

FIG. 5 shows the map of the interaction between the C-terminus of TCoA1and hSNF2H, hSNF2L, or NcoA-62/Skip.

FIG. 6 shows the map of the interaction between TCoA1 and NcoA-62/Skip.The minimal interacting region (position 224-317) is shown at thebottom.

FIG. 7 shows the map of the interaction between TCoA1 and hSNF2H. Theminimal interacting region (position 921-1017) is shown at the bottom.

FIG. 8 shows the proteins that associate in the interaction with TCoA1.Unverified interactions are shown in dashed lines.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be explained in detail below with referenceto examples, but it is not construed as being limited thereto.

EXAMPLE 1 Isolation of the “TCoA1” Gene

(1) Identification of a Novel Gene Comprising a Bromodomain

EST database was BLAST searched using various nucleotide sequencesencoding known bromodomain motifs. As a result, several potentialbromodomain-gene-encoding ESTs were identified by the search usingnucleotide sequence of Tetrahymena thermophila HAT A1 gene (Brownwell etal., (1996) Cell 84:843-851). One of these ESTs, the fetal lung cDNAlibrary-derived EST (W17142), was discovered to provide a novel gene.

(2) Isolation of Full-Length Nucleotide Sequence

The cloning of full-length cDNA against ESTW17142 was done as follows.First the PCR primers nb15U (GGATTATGAGGGGTTGAAGAGGG/SEQ ID NO:3) andnb15L (AAGGCAACAGAGTCTGTAGCCCAA/SEQ ID NO:4) were designed and a 119 bpamplification product was obtained by the polymerase chain reactionusing testicular cDNA as the template. The amplified product wasdirectly purified by a QIAquick (Qiagen) purifying column. Next, thetesticular cDNA library (HL3024a, Clontech) was screened using thisamplification product as the probe, and a re-screening of the librarywas done using the cDNA clone comprising the above-mentioned ESTsequence. The above probe was [α-³²P]dCTP labeled by random priming andpurified by CHROMA SPIN 10 column (Clontech). The library-filter washybridized using ExpressHyb Hybridization Solution (Clontech) for onehour at 65° C. The filter was washed at 65° C. with 0.5×SSC. 0.1% SDSuntil it reached the final stringency. Next, in order to identify thehybridizing clone, autoradiography was performed at −70° C. for one tothree days. The same procedure was done repeatedly until the obtainedclones were linked to acquire a nucleotide sequence covering the wholecoding-regions of the gene. All nucleotide sequences were determined bythe ABI377 Auto Sequencer using ABI dye-terminator chemistry. Sinceclones of the 5′ terminus were high in GC content, subdloning to theplasmid was done prior to sequence determination.

The library-screening gave 9865 bp nucleotide sequence. In this wholenucleotide sequence, an open reading frame (ORF) existed, which encoded2993 amino acids terminating at nucleotide position 8979. This ORF wasfollowed by 3′UTR of 877 bp until the polyA tail (FIG. 1). This sequenceis believed to be the whole sequence since, the length of the sequenceis comparable to the 10.5 Kb shown by northern blot analysis, and sincethe 5′ terminus is GC rich and coincides with the existence of a CpGisland seen at initiation points of many genes (Cross et al. (1995)Curr. Opin. Genet. Dev. 5:309-314). The nucleotide sequence of isolatedcDNA is shown in SEQ ID NO:2 and the amino acid sequence of the proteinencoded by said cDNA in SEQ ID NO: 1.

(3) Determination of Homology and the Motif Characteristics of theTranscriptional Factor

The motif was searched by PROSITE. The comparisons of proteins were doneusing Bestfit within GCG. The nuclear localization signal was identifiedby PSORT. Motif search revealed that several conserved regions anddomains were located in the amino acid sequence of presumed proteins(FIG. 1). These conserved regions had the C4HC3 zinc-finger (Aasland etal. (1995) Trends Biochem. Sci. 20:56-59; Koken et al. (1995) CR Acad.Sci. III, 318:733-739), a basic helix-loop-helix domain (Murre et al.(1989) Cell 58:537-544), an extensive hydrophobic glutamine-rich domain,CH2CH3 zinc-finger, and a bromodomain. Furthermore, there is a LXXLLmotif (Torchia et al. (1997) Nature 387:677-684; Heery et al. (1997)Nature 387:733-736) that most likely furnishes the interaction withnuclear receptors. All these motifs have the characteristic to presentthe functions as a transcriptional regulatory factor. As a result of thePSORT program, in all, eight consensus sequences were discovered at thenuclear site, which closely associate with the above function (Robbinset al. (1991) Cell 64:615-23). Expressing the function of the gene, itwas named “TCoA1” (transcriptional co-activator).

When the nucleotide sequence of “TCoA1” is analyzed upon thenon-redundant DNA database, it was found that “TCoA1” has a 100%homology with 2,183 bp of the FAC1 gene (Zhu et al. (1996) Biochemica etBiophysica Acta 1309:5-8) presumed to encode a protein of 810 residues.FAC1 was initially isolated by immunoscreening of an expression libraryusing Alz50 (Bowser et al. (1995) Dev. Neuroscience 17:20-37) monoclonalantibody. In addition to having a region that coincides spanning anextensive region with the nucleotide sequence of “TCoA1”, FAC1 alsocoincides with the “TCoA1” results, which were obtained using theexternal nucleotide sequence of the region that overlaps with FAC 1, inthe transcription size (Bowser et al. (1995) Dev. Neuroscience 17:20-37)and localization (Zhu et al. (1996) Biochemica et Biophysica Acta1309:5-8). In other words, it can be envisaged that the 2673 bpnucleotide sequence of FAC1 is a partial sequence that is equivalent tothe nucleotides from nucleotide position 248 of the 5′ terminus' tonucleotide position 2631. Comparison of the nucleotide sequences of FCA1and TCoA1 revealed that a single nucleotide-deleted error sequence (atposition 2400 A) exists in FAC1, and thus, it can be assumed thattranslation terminates at an early stage together with the shift of thereading frame of ORF. Similarly, a misrecognition of the initiationpoint of methionine residue had been triggered by a 5′ terminus sequenceerror in FAC 1.

The predicted amino acid sequence of “TCoA1” has several extensiveregions that have homologies with the presumed proteins of nematode (C.elegans), F26H11.2, F26H11.3a and F26H11.3b (Wilson at al., (1994)Nature 368:32-38). Results of analysis using “Gene Finder” software madethe prediction of the gene that encodes these proteins possible bysearching the genomic sequences contained in the F26H11 cosmid. Thenucleotide sequences of “TCoA1 ” N terminus coincided with F26HI 1 .gand C terminus with F26H11.I. This result showed that the both proteinspresumed by “TCoA1” and FCA1 are equivalent to a single protein in thenematode, and it is believed that “TCoA1” is the human homologue of thenematode protein.

EXAMPLE 2 Chromosome Mapping of “TCoA1”

To determine the chromosomal location of “TCoA1”, DNA obtainable fromeach of the 24 monochromosomal human/rodent somatic cell lines (Duboiset al. (1993) Genomics 16:315-319) acquired from Coriell CellRepositories, New Jersey, were amplified using the PCR primers nb15G(CCTCAGCTGCAACAAGTCC/SEQ ID NO:5) and nb15H (GCACTGCTTTGCTGAATTTGGA/SEQID NO:6). As predicted, 133 bp PCR product was amplified from the GM10567 cell system suggesting the possibility that the gene of theinvention is located on human chromosome no. 17 (FIG. 2A).

The “TCoA1” region locus was determined using Genebridge4 radiationhybrid panel of 91 hybrids (Walter et al. (1994) Nature Genetics7:22-28). Screening was done by re-using primer-G and primer-H andperforming PCR for that hybrid panel. By evaluating the respectivehybrids as being positive or negative in regard to amplification, thebinary code produced was compared with the similarity code for themarker that forms the framework map using the server at the web-addresshttp://www-genome.wi.mit.edu/cgi-bin/contig/rhmaiper.pl to determine thechromosomal location of the gene of the invention. “TCoA1” recognized tobe located in the marker D17S1557 (FIG. 2B). Only a score below 11showing the possibility of “TCoA1” existing at a site away from D17S1557was detected. This site coincides with the results by FISH showing FAC1is on chromosome no. 17 q24 (Bowser (1996) Genomics 38:455-457).

To find out a more precise location of “TCoA1”, screening byhierarchical PCR (Jones et al. (1994) Genomics 24:266-275) using theCEPH mega-YAC library and primers nb15S (AAGATGTTGTCTTGGAGCCGT/SEQ IDNO:7) and nb15Q (TTTTTTACCATTTGCTTCAGTCCC/SEQ ID NO:8). The single clone983d12 was identified but no information of this clone was obtainableeven by searching the map information of YAC 983d12 using CEPH infoclonedatabase (www.cephb.fr/infoclone.html). However, hybridization ofAlu-PCR products showed that the two clones (902c10 and 938f7) whichpartially overlap with 983d12, were both positive against D17S789 at theend of D17S1557. This coincides with the results of radiation hybridobtained by the Inventors and from a cytogenetic point-of-view, meansthat “TCoA1” is located on chromosome no. 17 q23 (Collins et al. (1996)Proc. Natl. Acad. Sci. USA 93:14771-14775). Though slightly different,this location is close to the chromosome no. 17 q24 (Bowser (1996)Genomics 38:455-457) location reported for FAC1.

EXAMPLE 3 Analysis of “TCoA1” Expression

Northern hybridization was done using 240 bp cDNA probe and 16 normaltissues as panels. The probe was [α-³²P]dCTP labeled by random primingand purified by CHROMA SPIN 10 column (Clontech). Hybridization fornorthern analysis was done using ExpressHyb Hybridization Solution(Clontech) for one hour at 65° C. The filter was washed at 65° C. with0.5×SSC, 0.1% SDS until it reached the final stringency. Next, in orderto identify hybridizing transcripts, autoradiography was performed at−70° C. for one to three days. mRNA blot was purchased from Clontech,and hybridization was done using the 240 bp cDNA probe equivalent to thenucleotide position 300-450. Approximately 10.5 kb mRNA was detected inalmost all tissues, and the size of the transcripts was equivalent tothat of ORF identified by the nucleotide sequence, and also coincidedwith the reported results for FACI (FIG. 3).

EXAMPLE 4 Determination of the Full-Length cDNA Nucleotide Sequence ofTCoA1

To obtain a complete cDNA, the Inventors screened the testicular cDNAlibrary (HL3024a, Clontech) again using the 119 bp amplification productof Example 1 (2) as the probe. Screening was done under the sameconditions as Example 1 (2).

When the cDNA nucleotide sequence obtained by the above screening wasread, it was a sequence of 9700 nucleotides in which an inframe stopcodon existed upstream the methionine initiation codon. Thus, theobtained cDNA was revealed to be full-length. The nucleotide sequence ofthe isolated full-length cDNA is given in SEQ ID NO:9, and the aminoacid sequence of the protein encoded by said cDNA in SEQ ID NO:10.

When the nucleotide sequence of TCoAl was compared with FAC1 (Zhu et al.(1996) Biochemica et Biophysica Acta 1309:5-8), the following nucleotidesequences coincided almost fully: position 57-1519 of FAC-1 withposition 461-1917 of TcoA1, and position 1898-2622 of FAC-1 withposition 1918-2643 of TCoA1. However, the position 1520-1897 of FAC-1does not exist in the nucleotide sequence of TCoA1. The nucleotidesequence of TCoA1 has an open reading frame (ORF) coding 2781 aminoacids, whereas the nucleotide sequence of FAC-1 has an ORF equivalent toa mere 810 amino acids, which is only a small part of TCoA1 beginningwith a methionine initiation codon. The amino acid sequence of TCoA1maintains two C4HC3 zinc-fingers (amino acid position 254-295) and onebromodomain (amino acid position 2684-2747). There is also an extensiveglutamine-rich region (amino acid position 1840-2400).

EXAMPLE 5 Identification of Proteins Interacting with the N TerminalRegion of TCoA1

Using a CDNA clone encoding the first 482 amino acids of TcoA1 includingthe C4HC3 zinc finger, yeast two-hybrid cDNA library (Clontech, PaloAlto) of the mouse-testis and human-brain was screened. This yeasttwo-hybrid cDNA library screening was done using yeast-vector PJ69-4A(James et al. (1996) Genetics 144(4):1425-36) according to the protocolof Clontech.

As a result, hSNF2H gene (Aihara et al. (1998) Cytogenet Cell Genet81(3-4):191-3) was isolated from the human cDNA library, and thecorresponding gene was isolated from the mouse cDNA library. hSNF2L gene(Aihara et al. (1998) Cytogenet Cell Genet 81(3-4):191-3) and also, atranscriptional co-activator NCoA-62 (also known as Skip) (Baudino etal. (1998) J. Biol. Chem. 273(26):16434-41, Dahl et al. (1998) Oncogene16(12):1579-86) were isolated from the human cDNA library.

hSNF2H/2L is the human homologue of D. melanogaster's ISWI. This ISWIprotein has been discovered within the chromatin reconstruction complexand this complex has been reported to be the molecular-device thatreconstructs nucleosomes upon DNA in an ATPase-dependent manner(Varga-Weisz et al. (1998) Curr. Opin. Cell Biol 10(3):346-53). Withinthese complexes, hSNF2H and hSNF2L acts as an ATPase subunit.

Recently, there was a report suggesting the possibility that ISWI alonehas an activity to reconstruct chromosomes (Corona et al. (1999) Mol.Cell 3(2):239-45). A 50 amino acid deletion at the C terminus was foundwhen the obtained full-length sequence of hSNF2H was compared with thesequence on the database (GenBank Accession No.AB010882) and alternativesplicing is believed to be occurring.

NCoA-62/Skip is a transcriptional co-activator interacting with Ski, aviral oncoprotein and ligand-binding domains of various nuclearreceptors (VDR, RAR). NCoA-62/Skip also has a homology with the fruitfly (Drosophila) Bx42 protein induced by ecdysone.

To verify the interactions between TCoA1 and above-mentioned proteins,analysis was done using constructs of mammalian two-hybrid system(Clontech, Palo Alto) according to protocols of Clontech. As a result,though a specific interaction could be found between TCoA1 and hSNF2H(FIG. 4), no interaction was seen for hSNF2L and Skip. Judging by thesimilarity of hSNF2H and hSNF2L, the lack of hSNF2L interaction wassurprising and hSNF2L was probably not expressed in this system.

EXAMPLE 6 The Interaction Map of TCoA1

The experiment for the construction of the interaction map was done asfollows, using the yeast two-hybrid system. cDNAs encoding variousregions (refer FIG. 5) were cloned to pAS vector (Clontech). Also cDNAsencoding the three proteins (hSNF2H, hSNF2L and NCoA-62/Skip) used inthe detection of interaction with various regions of TCA1, were clonedto the pACT vector (Clontech). A combination of these vectors wereintroduced to a yeast-host (PJ69-4A), and the interaction betweenproteins expressed within said host was detected using luciferase as thereporter.

The results revealed a region that interacts with all three proteins(hSNF2H, hSNF2L is and bx42 (NCoA-62/Skip)). Namely, as seen in FIG. 5,all three proteins interacted with the 85-247 amino acids of TCoA1.

This fact revealed that C4HC3 zinc finger known to be a proteininteracting site was omitted from the site interacting with these 3proteins.

EXAMPLE 7 Functional Analysis of the Bromodomain Interacting Protein

The clones (hSNF2H, hSNF2L, NCoA-62/Skip) interacting with TCoA1identified by the yeast two-hybrid screening, encode a huge polypeptide.Accordingly, the Inventors next identified the regions within theseproteins that interact with TCoA1 , using the yeast two-hybrid system.Specifically, a pACT vector (Clontech) constructs (FIG. 6, FIG. 7),which contained cDNA encoding a series of partially overlappingpolypeptides within NcoA-62 and hSNF2H, were prepared and introduced toyeast cells (PJ69-4A) together with the pAS vector (Clontech) containingcDNA encoding the amino acids of the 1-525 site of TCoA1 protein, andthe interaction between proteins expressed within said host was detectedusing luciferase as the reporter.

For NcoA-62, the region of approximately 450 amino acids including thecomplete carboxyl terminus domain of the original clone, and the seriesof five deletion clones in the said region were examined (FIG. 5). As aresult, amino acids of the position 224-317 within NcoA-62 wereidentified as the region interacting with TCoA1.

As for hSNF2H, the three deletion clones were analyzed (FIG. 7). As aresult, the region within hSNF2H that interacts with TCoA1 protein wasmapped to the carboxyl terminus (position 921-1017). The other clonehaving the same region (position 855-1017) failed to show anyinteraction. This may be due to the fact that this clone makes a specialsecondary structure.

INDUSTRIAL APPLICABILITY

The transcriptional regulatory factor and the DNA encoding said factorcan be used for the treatment of cancer and othercell-proliferation-linked diseases and also for the screening ofdrug-candidate compounds. Furthermore, antibodies binding to thetranscriptional regulatory factor of the present invention, compoundsthat regulate the function of said transcriptional regulatory factor,and compounds that inhibit the interaction between said transcriptionalregulatory factor and other proteins, may be utilized as therapeuticagents and preventive drugs for these diseases.

1-31. (canceled)
 32. An isolated antibody that specifically binds to apolypeptide the sequence of which consists of SEQ ID NO: 1 or
 10. 33.The antibody of claim 32, wherein the antibody is polyclonal.
 34. Theantibody of claim 32, wherein the antibody is monoclonal.
 35. Theantibody of claim 32, wherein the antibody is a human antibody.
 36. Theantibody of claim 32, wherein the antibody is a humanized antibody. 37.The antibody of claim 32, wherein the antibody is an IgG.
 38. Theantibody of claim 32, wherein the antibody is an IgM.
 39. An isolatedantibody that specifically binds to a polypeptide the sequence of whichconsists of SEQ ID NO:1 or 10 with up to 50 conservative amino acidsubstitutions.
 40. The antibody of claim 39, wherein the number ofconservative amino acid substitutions is up to
 30. 41. The antibody ofclaim 39, wherein the number of conservative amino acid substitutions isup to
 10. 42. The antibody of claim 39, wherein the number ofconservative amino acid substitutions is up to
 3. 43. The antibody ofclaim 39, wherein the antibody is polyclonal.
 44. The antibody of claim39, wherein the antibody is monoclonal.
 45. The antibody of claim 39,wherein the antibody is a human antibody.
 46. The antibody of claim 39,wherein the antibody is a humanized antibody.
 47. The antibody of claim39, wherein the antibody is an IgG.
 48. The antibody of claim 39,wherein the antibody is an IgM.